ABSTRACT Acute myeloid leukemia (AML) develops from dysregulated differentiation and self-renewal programs in the hematopoietic stem cells (HSCs) or early progenitors. The leukemia fusion protein CBF?-SMMHC represses the transcription regulatory program of the core-binding factor RUNX1/CBF? and generates preleukemic myeloid progenitor cells. These cells are susceptible to induce myeloid leukemia in cooperation with other oncogenic mutations. However, the pathways targeted by this fusion protein to induce pre-leukemic blasts, and the pathways involved in the oncogenic switch to leukemia are not known. We have developed a small molecule inhibitor of CBF?-SMMHC activity that binds to the fusion protein with high specificity, and disrupts CBF?-SMMHC/RUNX1 binding. As a result, RUNX1/CBF? dimers increases their occupancy at the promoters of target genes and induces the transcription regulation of RUNX1/CBFB target genes Preliminary studies show that pharmacologic CBF?-SMMHC inhibition induces apoptosis in inv(16) AML cells but not to other AML types or normal hematopoietic progenitors, suggesting an oncogene addiction switch. We hypothesize that CBF?-SMMHC blocks the differentiation of preleukemic cells by disrupting expression of CBF targets, and that this event is reversible. However, the progression from preleukemia to leukemia is based on transcriptional and chromatin changes directed by CBF?-SMMHC-mediated regulation of RUNX1/CBF? activity. In addition, we will evaluate the hypothesis that this switch is due to the upregulation of MYC expression, and that in leukemic cells (but not in preleukemic cells) MYC activity maintains the survival programs. This hypothesis will be testes in the following three specific aims: Specific Aim 1. Determine how CBF?-SMMHC induces MYC expression and maintains AML survival. Specific Aim 2. Elucidate the transcription and chromatin modification directed by CBF?-SMMHC. Specific Aim 3. Determine the targets of CBF?-SMMHC mediated pre-leukemia and leukemia initiation. In summary, the proposed studies combine our expertise in core binding factor leukemia, our mouse models for inv(16) AML and our novel pharmacologic inhibitor to investigate the mechanism of CBF?-SMMHC directed preleukemia and the oncogene addiction in leukemia initiation and maintenance.